ES2760249T3 - Combined pharmaceutical composition - Google Patents

Abstract

Combined pharmaceutical composition comprising: a) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin or derivatives thereof having at least 10% of the activity of human IL-15 on induction of proliferation from the kit225 cell line, and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to human IL-15, a polynucleotide encoding it, or a vector comprising such a polynucleotide; and b) an antibody that antagonizes an immune pathway involved in the inhibition of T-cell activation, or a fragment thereof that can react with the same antigen as its antibody counterpart, a polynucleotide encoding it, or a vector that it comprises such a polynucleotide, wherein said antibody is a PD-1 / PD-L1 / PD-L2 antagonist, wherein said antibody antagonizes either by binding to the PD-1 receptor or by binding of the PD-L1 or PD-L2 ligand; wherein said conjugate and the antibody or fragment thereof are not linked.

Description

DESCRIPTION

Combined pharmaceutical composition

The present invention relates to a new "combined pharmaceutical composition", and more specifically to a combination for treating cancer of a specific IL-15 superagonist and of an antibody that antagonizes an immune pathway involved in inhibiting T-cell activation as defined in the claims.

Background

An adaptive immune response involves clonal activation, selection, and proliferation of two main classes of lymphocytes called T cells and B cells. After encountering an antigen, T cells proliferate and differentiate into antigen-specific effector cells, while B cells they proliferate and differentiate into cells that secrete antibodies.

T cell activation is a multi-stage process that requires several signaling events between the T cell and an antigen presenting cell (APC). For T cell activation to occur, two types of signals must be delivered to a resting T cell.

The first type is mediated by the antigen-specific T-cell receptor (TcR), and confers specificity to the immune response.

The second costimulatory type regulates the magnitude of the response and is administered through accessory receptors on the T cell. These receptors comprise immunosuppressive receptors (for example CTL-A4, PD-1 or KIR inhibitors) and costimulatory receptors (for example CD40, 4-1BB, OX-40 or glucocorticoid-induced TNFR-related protein (GITR)).

Therapeutic strategies have been developed on the inhibition of immunosuppressive receptors or on the activation of costimulatory receptors to enhance the anti-tumor response of the immune system.

WO 2012/175222 discloses an immunocytokine comprising (a) a conjugate and (b) an antibody or a fragment thereof linked directly or indirectly by covalence to said conjugate, wherein said conjugate comprises (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof, and a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof. Xu et al. (Cancer Research, vol. 73, No. 10, pages 3075-3086, 2013) disclose that a single dose of a complex of an interleukin (IL) -15 superagonist mutant and aSu / Fc fusion protein of Dimeric IL-15 receptor, but not IL-15 alone, kills 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice.

In fact, combined strategies are also envisaged. Now the results are very dangerous depending on the compounds tested.

Summary of the invention

Now, the inventors show that a combination of its specific compound, called RLI, with an antibody that antagonizes an immune pathway involved in inhibiting T-cell activation results in a very high percentage of tumor remission, while such remission could not be contemplated in view of tumor remission obtained with RLI or immunosuppressive receptor antagonists alone.

Furthermore, this synergistic action was also obtained with a low dose combination in which a low dose of RLI was combined with a low dose of anti-PD1. Surprisingly, and compared to the previous combination, this combination has provided strong and synergistic inhibition of tumor growth.

This strong synergy allows us to contemplate new therapies.

Accordingly, the present invention relates to a combined pharmaceutical composition comprising:

1) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof having at least 10% of the activity of human IL-15 on the induction of proliferation of the kit225 cell line , and ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to human IL-15; a polynucleotide encoding therefor, or a vector comprising such a polynucleotide; and

2) an antibody that antagonizes an immune pathway involved in inhibiting T-cell activation, or a fragment thereof that can react with the same antigen as its antibody counterpart, a polynucleotide encoding therefor, or a vector comprising such a polynucleotide, wherein said antibody is a PD-1 / PD-L1 / PD-L2 antagonist, wherein said antibody antagonizes either by binding of the PD-1 receptor or by binding of the PD-L1 or PD-L2 ligand, in which said conjugate and the antibody or fragment thereof are not linked.

In a second aspect, the invention relates to a pharmaceutical composition for use in treating cancer in a subject comprising

(A) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof having at least 10% of the activity of human IL-15 on induction of cell line proliferation kit225, and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to IL-15 human, a polynucleotide encoding therefor, or a vector comprising such a polynucleotide, wherein the use comprises the simultaneous, separate or sequential administration of (a) said pharmaceutical composition and (b) an antibody that antagonizes an immune pathway involved in inhibiting T-cell activation, or a fragment thereof that can react with the same antigen as its antibody counterpart, a polynucleotide encoding it, or a vector that comprises a polynucl such an eotide, or (B) an antibody that antagonizes an immune pathway involved in inhibiting T-cell activation, or a fragment thereof that can react with the same antigen as its antibody counterpart, a polynucleotide that encodes the same, or a vector comprising such a polynucleotide, wherein the use comprises the simultaneous, separate or sequential administration of (a) said pharmaceutical composition and (b) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof having at least 10% of the activity of human IL-15 on the induction of proliferation of the cell line kit225, and ii) a polypeptide comprising the amino acid sequence of the Sushi domain of IL-15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to human IL-15, a polynucleotide encoding it, or a vector that it comprises such a polynucleotide, and

wherein said antibody is a PD-1 / PD-L1 / PD-L2 antagonist,

wherein said antibody antagonizes either by binding to the PD-1 receptor or by binding to the PD-L1 or PD-L2 ligand.

The pharmaceutical composition for use in the treatment of cancer or the combined pharmaceutical composition of the present invention are preferably adapted for simultaneous, separate or sequential administration for use in treating cancer in a subject.

Brief description of the drawings

Figure 1 shows the injection protocol used in the BIOXELL anti-PD1 mouse cancer model. Figure 2 shows the presence of TIL in the CT26 model.

Figure 3 shows the combination anti-PD1 (RPMI1-14) and RLI therapy in mice.

Figure 4 shows the survival of the mice for the anti-PD1 / RLI combination treatment.

Figure 5 shows the injection protocol used in the mouse cancer model.

Figure 6 shows the combination anti-PD1 (mBAT) and RLI therapy in mice.

Detailed description

Conjugate

The term "interleukin 15" has its general meaning in the art and refers to a cytokine with structural similarity to IL-2 (GRABSTEIN et al., Science, vol. 264 (5161), pp .: 965-968, 1994) . This cytokine is also known as IL-15, IL15, or MGC9721. This cytokine and IL-2 share many biological activities and were found to bind to common hematopoietin receptor subunits. Therefore, they can compete for the same receptor, negatively regulating the activity of the other. It has been established that IL-15 regulates the activation and proliferation of T cells and natural cytolytic lymphocytes, and that the number of CD8 + memory cells is shown to be controlled by a balance between this cytokine and IL2. IL-15 activity can be measured by determining its induction of proliferation on the kit225 cell line (HORI et al., Blood, vol. 70 (4), pp .: 1069-72, 1987), as disclosed in the examples.

Said IL-15 or derivatives thereof have at least 10% of the activity of human interleukin-15 on the induction of proliferation of the kit225 cell line, preferably at least 25% and more preferably at least 50%.

Said interleukin 15 is a mammalian interleukin 15, preferably a primate interleukin 15, and more preferably a human interleukin 15.

The skilled person can easily identify the mammalian interleukin 15. As an example, interleukin 15 can be cited from Sus scrofa (registration number ABF82250), from Rattus norvegicus (registration number NP_037261), from Mus musculus (registration number NP_032383), from Bos taurus (registration number NP_776515), from Oryctolagus cuniculus (registration number NP_001075685), from Ovies aries (registration number NP_001009734), from Felis catus (registration number NP_001009207), from Macaca fascicularis (registration number BAA19149), from Homo sapiens (registration number NP_000576), from Macaca mulatta (registration number NP_001038196), Cavia porcellus (registration number NP_001166300) or Clorocebus sabaeus (registration number ACI289).

As used herein, the term "mammalian interleukin 15" refers to the consensus sequence SEQ ID NO: 1.

The expert can easily identify primate interleukin 15. As an example, interleukin 15 from Sus scrofa (registration number ABF82250), from Oryctolagus cuniculus (registration number NP_001075685), from Macaca fascicularis (registration number BAA19149), from Homo sapiens (registration number NP_000576), from Macaca can be cited. mulatta (registration number NP_001038196) or Clorocebus sabaeus (registration number ACI289).

As used herein, the term "primate interleukin 15" refers to the consensus sequence SEQ ID NO: 2.

The expert can easily identify human interleukin 15 and refer to the amino acid sequence SEQ ID NO: 3.

As used herein, the term "interleukin 15 derivatives" refers to an amino acid sequence that has a percent identity of at least 92.5% (ie corresponding to approximately 10 amino acid substitutions) with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SeQ ID NO: 3, preferably at least 96% (i.e. corresponding to approximately 5 amino acid substitutions), and more preferably at least 98.5% (ie, corresponding to about 2 amino acid substitutions) or at least 99% (ie, corresponding to about 1 amino acid substitution). The expert can easily identify such derivatives in view of his personal knowledge and the teaching of the present patent application. As an example of such derivatives, those described in PCT international patent application WO 2009/135031 can be cited. It will also be understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids increase the half-life of the polypeptide.

As used herein, "percent identity" between two amino acid sequences means the percentage of identical amino acids, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being spread randomly over the amino acid sequences. As used herein, "best alignment" or "optimal alignment" means the alignment for which the determined percent identity (see below) is the highest. Sequence comparison between two amino acid sequences is usually performed by comparing these sequences that have been previously aligned for best alignment; This comparison is done in comparison segments in order to identify and compare local regions of similarity. The best sequence alignment for comparison can be performed, in addition to manually, using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., Vol. 2, pp .: 482, 1981), using the local homology algorithm developed by NEDDLEMAN and WUNSCH (J. Mol. Biol., vol. 48, p .: 443, 1970), using the similarity method developed by PEARSON and LIPMAN (Proc. Natl. Acd. Sci. USA, vol. 85, pg: 2444, 1988), using computer software using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), using MUSCLE multiple alignment algorithms (Edgar, Robert C., Nucleic Acids Research, vol. 32, pp .: 1792, 2004). To obtain the best local alignment, the BLAST software can preferably be used with the BLOSUM 62 matrix. The percent identity between two amino acid sequences is determined by comparing these two optimally aligned sequences, the amino acid sequences being able to encompass additions or deletions with respect to the reference sequence in order to obtain the optimal alignment between these two sequences. The identity percentage is calculated by determining the number of identical positions between these two sequences, and dividing this number by the total number of compared positions, and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

Preferably, the interleukin 15 derivatives are IL-15 agonists or super antagonists. A person skilled in the art can easily identify an IL-15 agonist or super-agonist. As an example of IL-15 agonist or superagonist, those disclosed in international patent application WO 2005/085282 or in ZHU et al. (J. Immunol., Vol. 183 (6), pp .: 3598-607, 2009).

Still preferably, said IL-15 agonist or superagonist is selected from the group comprising / consisting of L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y and N72P (referring to the sequence of IL- 15 human, SEQ ID NO: 3).

As used herein, the term "the IL-15Ra sushi domain" has its general meaning in the art and refers to a domain that begins at the first cysteine residue (C1) after the IL signal peptide -15Ra, and ending at the fourth cysteine residue (C4) after said signal peptide. Said sushi domain corresponding to a portion of the extracellular region of IL-15Ra is necessary for its binding to IL-15 (WEI et al., J. Immunol., Vol. 167 (1), pp .: 277-282, 2001 ).

Said sushi domain of IL-15Ra or derivatives thereof have at least 10% of the binding activity of the sushi domain of human IL-15Ra to human interleukin-15, preferably at least 25% and more preferably at least 50 %. Said binding activity can be easily determined by the method disclosed in WEI et al. (mentioned earlier, 2001).

Said sushi domain of IL-15Ra is the sushi domain of a mammalian IL-15Ra, preferably the sushi domain of a primate IL-15Ra and more preferably the sushi domain of human IL-15Ra.

The expert can easily identify the sushi domain of a mammalian IL-15Ra. As an example, the sushi domain of an IL-15Ra from Rattus norvegicus (registration number XP_002728555), from Mus musculus (registration number EDL08026), from Bos taurus (registration number XP_002692113), from Oryctolagus cuniculus (registration number) can be cited. XP_002723298), of Macaca fascicularis (registration number ACI42785), of Macaca nemestrina (registration number ACI42783), of Homo sapiens (registration number Q13261.1), of Macaca mulatta (registration number NP_001166315), Pongo abelii (registration number registration XP_002820541), Cercocebus torquatus (registration number ACI42784), Callithrix jacchus (registration number XP_002750073) or Cavia porcellus (registration number NP_001166314).

As used herein, the term "mammalian IL-15Ra sushi domain" refers to the consensus sequence SEQ ID NO: 4.

Preferably, the polypeptide comprising the amino acid sequence of the sushi domain of a mammalian IL-15Ra refers to the consensus sequence SEQ ID NO: 5.

The expert can easily identify the sushi domain of a primate IL-15Ra. As an example, sushi domains of IL-15Ra from Oryctolagus cuniculus, Macaca fascicularis, Macaca nemestrina, Homo sapiens, Macaca mulatta , Pongo abelii , Cercocebus torquatus or Callithrix jacchus can be mentioned .

As used herein, the term "sushi domain of a primate IL-15Ra" refers to the consensus sequence SEQ ID NO: 6.

Preferably, the polypeptide comprising the amino acid sequence of the sushi domain of a primate IL-15Ra refers to the consensus sequence SEQ ID NO: 7.

The expert can easily identify the sushi domain of human IL-15Ra and refer to the amino acid sequence SEQ ID NO: 8.

Preferably, the polypeptide comprising the amino acid sequence of the sushi domain of human IL-15Ra refers to SEQ ID NO: 9.

As used herein, the term "derivatives of the IL-15Ra sushi domain" refers to an amino acid sequence that has a percent identity of at least 92% (ie corresponding to approximately 5 substitutions amino acids) with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 , preferably at least 96% (ie, corresponding to about 2 amino acid substitutions), and more preferably at least 98% (ie, corresponding to about 1 amino acid substitution). Such derivatives comprise the four cysteine residues of the L-15Ra sushi domain and the expert You can easily identify them in light of your general knowledge and teaching of the present patent application. It will also be understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids allow to increase the half-life of the polypeptide.

According to a preferred embodiment, the conjugate comprises (ii) a polypeptide comprising the amino acid sequence of the sushi and hinge domains of IL-15Ra or derivatives thereof.

The hinge domain of IL-15Ra is defined as the amino acid sequence that begins at the first amino residue after the sushi domain and ends at the last amino acid residue before the first possible glycosylation site. In human IL-15Ra, the amino acid sequence of the hinge region consists of the fourteen amino acids that are located after the sushi domain of this IL-15Ralpha, in a C-terminal position in relation to said sushi domain, that is, said region IL-15Ralfa hinge starts at the first amino acid after said cysteine residue (C4), and ends at the fourteenth amino acid (counting in the conventional orientation "from N-terminal to C-terminal").

Such sushi and hinge domains of IL-15Ra are the sushi and hinge domains of a mammalian IL-15Ra, preferably the sushi and hinge domains of a primate IL-15Ra and more preferably the sushi and hinge domains of human IL-15Ra .

The skilled person can easily identify the amino acid sequence of the sushi and hinge domains of a mammalian IL-15Ra. As used herein, the term "sushi and hinge domains of a mammalian IL-15Ra" refers to the consensus sequence SEQ ID NO: 10.

The skilled person can easily identify the amino acid sequence of the sushi and hinge domains of a primate IL-15Ra. As used herein, the term "sushi and hinge domains of a primate IL-15Ra" refers to the consensus sequence SEQ ID NO: 11.

The skilled person can easily identify the amino acid sequence of the sushi and hinge domains of human IL-15Ra. As used herein, the term "sushi and hinge domains of human IL-15Ra" refers to the consensus sequence SEQ ID NO: 12.

As used herein, the term "derivatives of the sushi and hinge domains of IL-15Ra" refers to an amino acid sequence having a percent identity of at least 93% (ie corresponding to approximately 5 amino acid substitutions) with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, preferably at least 97% (i.e. corresponding to approximately 2 amino acid substitutions), and more preferably at least 98% (ie, corresponding to about 1 amino acid substitution). Such derivatives comprise the four cysteine residues of the L-15Ra sushi domain and can be easily identified by the skilled person in view of his general knowledge and teaching of the present patent application. It will also be understood that natural amino acids can be replaced by chemically modified amino acids. Typically, such chemically modified amino acids allow to increase the half-life of the polypeptide.

Both polypeptides i) and ii) of the conjugate can be non-covalently linked such as in the complex disclosed in US Patent 8,124,084 ^b 2. Said conjugate or complex can be easily obtained by providing a suitable amount of the polypeptide i) , providing a suitable amount of the polypeptide ii), mixing both polypeptides under suitable ionic and pH conditions for a duration sufficient to allow the formation of the complex (that is, conjugate), and optionally concentrating or purifying said complex. Complex (ie, conjugate) polypeptides can be formed, for example, using a peptide synthesizer according to conventional methods; expressing each polypeptide separately in a cell or cell extract, then isolating and purifying the polypeptide. Optionally, the therapeutic polypeptide complex of the invention can be formed by expressing both polypeptides i) and ii) in the same cell or cell extract, then isolating and purifying the complexes, for example, using chromatographic techniques, such as affinity chromatography with antibodies against lymphokine portion, the lymphokine receptor portion, or against the complex.

Both polypeptides i) and ii) of the conjugate can also be covalently linked using bifunctional protein coupling agents or on a fusion protein.

Bifunctional protein coupling agents as well as methods using them are well known to the skilled artisan, and include, as examples, N-succinimidyl (2-pyridyldithio) propionate (SPDP), (N-maleimidomethyl) cyclohexan-1-carboxylate from succinimidyl, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexandiamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-fluorine active compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

The term "fusion protein" refers to a protein created through the joining of two or more genes that originally encoded different proteins. Also known as a chimeric protein. Translation of this fusion gene results in a single polypeptide with functional properties that are derived from each of the original proteins. Recombinant fusion proteins are artificially created using recombinant DNA technology for use in biological or therapeutic research. A recombinant fusion protein is a protein created through genetic engineering modification of a fusion gene. This typically involves removing the stop codon from a cDNA sequence encoding the first protein, then attaching the second protein cDNA sequence in-frame through extension PCR by ligation or ligation. That DNA sequence will then be expressed by a cell as the only protein. The protein can be genetically engineered to include the complete sequence of both original proteins, or just a portion of either.

In a preferred embodiment, the conjugate is a fusion protein.

The amino acid sequence of interleukin 15 or derivatives thereof may be in a C-terminal position or in an N-terminal position relative to the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof. Preferably, the amino acid sequence of interleukin 15 or derivatives thereof is in a C-terminal position relative to the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof.

The amino acid sequence of interleukin 15 or derivatives thereof and the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof can be separated by a first "linker" amino acid sequence. Said first "linker" amino acid sequence may be of sufficient length to ensure that the fusion protein forms suitable secondary and tertiary structures.

The length of the first linker amino acid sequence can be varied without significantly affecting the biological activity of the fusion protein. Typically, the first linker amino acid sequence comprises at least one, but not less than 30 amino acids eg, a linker of 2-30 amino acids, preferably 10-30 amino acids, more preferably 15-30 amino acids, even more preferably 15 -25 amino acids, most preferably 18-22 amino acids.

Preferred linker amino acid sequences are those that allow the conjugate to adopt a suitable conformation (ie, a conformation that allows adequate signal transduction activity via the IL-15Rbeta / gamma signaling pathway).

The first most suitable linker amino acid sequences (1) will adopt a flexible extended conformation, (2) will not have a propensity to develop an ordered secondary structure that can interact with the functional domains of fusion proteins, and (3) will be charged or Minimal hydrophobic that can promote interaction with functional protein domains.

Preferably, the first linker amino acid sequence comprises near neutral amino acids selected from the group comprising Gly (G), Asn (N), Ser (S), Thr (T), Ala (A), Leu (L) and Gln ( Q), most preferably in the group comprising Gly (G), Asn (N) and Ser (S).

Examples of linker sequences are described in US Patent Nos. 5,073,627 and 5,108,910.

Illustrative flexible linkers that are more particularly suitable for the present invention include those encoded by the sequences of SEQ ID NO: 13 (SGGSGGGGSGGGSGGGGGSLQ), SEQ ID NO: 14 (SGGSGGGGSGGGSGGGGSGG.) SEQ ID NO: 15 (SGGGSGGGGSGG).

Still preferably, the conjugate is a fusion protein having the sequence SEQ ID NO: 16 or SEQ ID NO: 17.

Immunosuppressive receptor antagonist antibody

The term "antibody" refers to an immunoglobulin molecule corresponding to a tetramer comprising four polypeptide chains, two identical heavy (H) chains (approximately 50-70 kDa when full length), and two identical (L) light chains ( approximately 25 kDa when full length) interconnected by disulfide bonds. Light chains are classified as kappa and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Each heavy chain is composed of an N-terminal heavy chain variable region (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3) for IgG, IgD, and IgA; and 4 domains (CH1, CH2, CH3, and CH4) for IgM and IgE. Each light chain is composed of one N-terminal light chain variable region (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is composed of a domain, CL. The HCVR and LCVR regions can be further subdivided into hypervariability regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Lattice Regions (FR). Each HCVR and LCVR is made up of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each domain is according to well-known conventions. The functional ability of the antibody to bind to a particular antigen depends on the variable regions of each light / heavy chain pair, and is largely determined by CDRs.

The term "antibody", as used herein, refers to a monoclonal antibody per se. A monoclonal antibody can be a human antibody, a chimeric antibody, and / or a humanized antibody.

Advantageously, the term antibody refers to an IgG, such as IgG1, IgG2 (IgG2a or IgG2b), IgG3, and IgG4. Preferably, the term antibody refers to IgG1 or IgG2, and more preferably IgG2a.

"Chimeric antibody" means an antibody that is composed of variable regions of a murine immunoglobulin and constant regions of a human immunoglobulin. This alteration is simply to replace the constant region of a human antibody with the murine constant region, thereby resulting in a human / murine chimera that may have low enough immunogenicity to be acceptable for pharmaceutical use. Various methods of producing such chimeric antibodies have been reported, thereby forming part of the general knowledge of one skilled in the art (see, eg, US Patent No. 5,225,539).

"Humanized antibody" means an antibody that is partially or completely composed of amino acid sequences derived from a germline of human antibody by altering the sequence of an antibody that has non-human complementarity determining regions (CDRs). This humanization of the variable region of the antibody and finally CDR is accomplished by techniques that are well known in the art. As an example, UK Patent Application GB 2188638A and US Patent No. 5,585,089 disclose processes in which recombinant antibodies are produced in which the only portion of the antibody to be replaced is the complementarity determining region, or "CDR". The CDR grafting technique has been used to generate antibodies consisting of murine CDRs and framework and framework regions of human variable regions (see, eg, RIECHMANN et al., Nature, vol. 332, p .: 323-327 , 1988). These antibodies maintain the human constant regions that are necessary for Fc-dependent effector function, but are much less likely to evoke an immune response against the antibody. As an example, the framework regions of the variable regions are replaced by corresponding human framework regions that leave the non-human CDR substantially intact, or even replace the CDR with sequences derived from a human genome. Fully human antibodies are produced in genetically modified mice whose immune systems have been altered to correspond to human immune systems. As mentioned above, it is sufficient for use in the methods of the invention to employ an immunologically specific fragment of the antibody, including fragments representing single chain forms.

Again, a humanized antibody refers to an antibody that comprises a human framework region, at least one CDR of a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e. , at least about 85 or 90%, preferably at least 95% identical. Therefore, all parts of a humanized antibody, except possibly CDRs, are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. For example, a humanized immunoglobulin would not normally encompass a human constant region / mouse variable region chimeric antibody. As an example, humanized immunoglobulin design can be carried out as follows: when an amino acid falls into the following category, the framework region amino acid of a human immunoglobulin to be used (acceptor immunoglobulin) is replaced by an amino acid of framework region of a non-human immunoglobulin providing CDR (donor immunoglobulin): (a) the amino acid in the framework region of human acceptor immunoglobulin is unusual for the human immunoglobulin at that position, whereas the corresponding amino acid in immunoglobulin donor is typical for human immunoglobulin in that position; (b) the amino acid position is immediately adjacent to one of the CDRs; or (c) any lattice region amino acid side chain atom is within approximately 5-6 Angstroms (center to center) of any CDR amino acid atom in a three-dimensional immunoglobulin model (CO MS et al., Proc. Natl. Acad. Sci. USA, vol. 88, pp .: 2869, 1991). When each of the amino acids in the human framework region of the acceptor immunoglobulin and a corresponding amino acid in the donor immunoglobulin is unusual for human immunoglobulin at that position, such amino acid is replaced by a typical amino acid for human immunoglobulin at that position.

The term "antibody fragment" as used herein refers to an antibody fragment that can react with the same antigen as its antibody counterpart. Such fragments can be easily identified by the skilled person and comprise, as an example, Fab fragment (eg, by digestion with papain), Fab 'fragment (eg, by pepsin digestion and partial reduction), F (ab') 2 fragment (eg, by pepsin digestion), Facb (eg, by plasmin digestion), Fd ( eg, by pepsin digestion, partial reduction and re-aggregation), and also scFv fragment (single chain Fv; eg, by molecular biology techniques) are encompassed by the invention.

Such fragments can be produced by recombinant, synthetic or enzymatic cleavage techniques, as known in the art and / or as described herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding an F (ab ') 2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. The various antibody portions can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetically engineered modification techniques.

Preferably, said antibody fragment is an scFv fragment.

The term "antibody that antagonizes an immune pathway involved in the inhibition of T-cell activation" refers to an antibody that antagonizes the immunosuppressive receptor PD-1 either by binding to this receptor or by binding to this ligand, thereby promoting immune activation avoiding down-regulation signals. It is further disclosed that another "antibody that antagonizes an immune pathway involved in the inhibition of T-cell activation" is an antibody that antagonizes the immunosuppressive receptor CTL-A4 or KIR inhibitors either by binding to or by binding to this receptor ligand, thereby promoting immune activation by avoiding down-regulation signals. As an example of an immunosuppressive receptor antagonist antibody, antibodies corresponding to CTL-A4, PD-1 / PD-L1, PD-1 / PD-L2, KIR inhibitors, CD276, VTCN1, BTLA / HVEM, LAG3, HAVCR2 and ADORA2A antagonist, preferably PD-1 / PD-L1 antibodies.

CTL-A4 (cytotoxic lymphocyte-associated antigen, also called CD 152) was discovered in 1987 (BRUNET et al., Nature, vol. 328, pp .: 267-270, 1987). The role of CTL-A4 is primarily to inhibit T cell activation and this was demonstrated in CTL-A4 deficient mice suffering from massive lymphoproliferation (CHAMBERS et al., Immunity, vol. 7, pp .: 8855-8959, 1997) . Now, CTL-A4 blocking has been shown to enhance T cell responses in vitro (WALUNAS et al., Immunity, vol. 1, pp .: 405-413, 1994) and in vivo (KEARNEY, J. Immunol, vol.

155, pp .: 1032-1036, 1995) and also that it increases anti-tumor immunity (LEACH, Science, vol. 271, pp .: 1734-1736, 1996). As an example of antibodies corresponding to CTL-A4 antagonists, there may be cited ipilimumab (also called MDX-010 and 10D1, available from MEDAREX and marketed as YERVOY ™ by BRISTOL-MYERS SQUIBB COMPANY) disclosed in document W ^or 01/14424 , ticilimumab (also known as 11.2.1 and CP-675.206) disclosed in WO 00/37504, and also the CTL-A4 antibodies disclosed in international patent applications WO 98/42752, WO 01/14424 WO 2004/035607 and WO 2012/120125, in EP 1212422 and EP 1262193, in US Patent Nos. US 5,811,097, US 5,855,887, US 5,977,318, US 6,051,227, US 6,207. 156, US 6,682,736, US 6,984,720, US 7,109,003 and US 7,132,281.

Programmed cell death 1 also known as PD-1 (also called PDCD1 or CD279) is a ~ 55 kD type I membrane glycoprotein. PD-1 is a receptor on the CD28 costimulatory gene family, which is moderately expressed in natural cytolytic lymphocytes and undifferentiated T and B cells and up-regulated by T / B cell receptor signaling in lymphocytes, monocytes, and myeloid cells. PD-1 has two known ligands with different expression profiles, PD-L1 (B7-H1), which is widely expressed, that is, in undifferentiated lymphocytes in activated B and T cells, monocytes and dendritic cells, and PD-L2 ( B7-DC), whose expression is restricted, that is, in activated dendritic cells, macrophages and monocytes and in vascular endothelial cells. In several models of murine syngeneic tumors, blocking of either PD-1 or PD-L1 significantly inhibited tumor growth or induced complete remission. Therefore, PD-1 is recognized as an important member in the regulation of the immune system and the maintenance of peripheral tolerance. As an example of antibodies corresponding to PD-1 / PD-L1 / PD-L2 antagonists, there may be cited nivolumab (also known as BmS-936558 or MDX1106; anti-PD-1 antibody, BRISTOL-MYERS SQUIBB) is disclosed in WO 2006/121168, Merck 3745 (also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody) is disclosed in WO 2009/114335, CT-01 1 (also known as hBAT or hBAT-1, anti-PD-1 antibody) is disclosed in WO 2009/101611, lambrolizumab is disclosed in WO2008 / 156712, AMP514 is disclosed in WO2010 / 027423, WO2010 / 027827, WO2010 / 027828 and WO2010 / 098788, and also the antibodies disclosed in the international patent applications WO 2004/056875, WO 2006/056875, WO 2008/083174, WO 2010/029434, WO 2010/029435, WO 2010/036959, WO 2010/089411, WO 2011/110604, WO 2012/135408 and WO 2012/145493. Said PD-1 antagonist may correspond to an anti-PD-L1 antibody such as MDX-1 105 (also known as BMS-936559, anti-PD-L1 antibody) disclosed in WO 2007/005874 or YW243. 55.S70 (also known as MPDL3280A or RG7446; anti-PD-L1 antibody) disclosed in WO 2010/077634.

Cytolytic lymphocyte immunoglobulin-like receptors (KIRs) are a family of surface proteins which are found in important cells of the immune system called natural cytolytic lymphocytes (NK). They regulate the killing function of these cells by interacting with MHC class I molecules, which are expressed in all cell types. This interaction allows them to detect virus-infected cells or tumor cells that have a characteristic low level of MHC class I on their surface. Most KIRs are inhibitors, which means that their recognition of MHC suppresses the cytotoxic activity of their natural cytolytic lymphocyte. Only a limited number of KIRs have the ability to activate the cell.

Inhibitory KIRs have a long cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), which transduces inhibitory signals to the natural cytolytic lymphocyte upon coupling of its MHC class I ligands. Known inhibitory KIRs include members of the subfamilies KIR2DL and KIR3DL comprising KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3. As an example of antibodies corresponding to inhibitory KIR antagonists, there may be cited antibody 1-7F9 disclosed in WO 2006/003179.

Members of the B7 family of ligands are known to have strong immunoregulatory activity by immune cells and more recently by tumor cells, a family comprising CD276 and VTCN1.

CD276 (differentiation pool 276) (also known as B7H3, B7-H3; B7RP-2; 4Ig-B7-H3) is a type I transmembrane protein from the B7 family of ligands, first identified in dendritic cells and activated T cells. Some solid tumors express CD276 and are thought to participate in the regulation of the T cell-mediated immune response. More especially, CD276 appears to down-regulate T-helper immune response and suppress immunity (SUH WK et al., 2003. Nat Immunol 4 (9): 899-906). As an example of a CD276 antagonist, there may be cited the 8H9 antibody disclosed in patent application US 2005/0169932 and WO 2008/116219, and other antibodies such as MGA271 disclosed in WO 2011/109400.

VTCN1 (domain of set V containing T cell activation inhibitor 1), also known as B7X; B7H4; B7S1; B7-H4, is also a member of the B7 family. VTCN1 is thought to play a role in the inhibitory regulation of T-cell response, and studies have shown that high levels of this protein have been correlated with tumor progression. As an example of VTCN1 antagonists, the antibodies disclosed in WO 2009/073533 can be cited.

BTLA (B and T lymphocyte attenuator), also known as CD272, is induced during T cell activation, and remains expressed in Th1 cells but not in Th2 cells. BTLA exhibits inhibition of T cells through interaction with tumor necrosis factor (receptor), member 14 (TNFRSF14), also known as herpes virus (HVEM) mediator of entry, TR2; TIE; HVEA; CD270; LIGHTR. TNFRSF14 was identified as a cellular mediator of herpes simplex virus (HSV) entry. The cytoplasmic region of this receptor was found to bind to several members of the TRAF family, which can mediate signal transduction pathways that activate the immune response. Finally, BTLA / HVEM complexes down-regulate T cell immune responses. As an example of BTLA / HVEM antagonist, the antibodies disclosed in WO 2008/076560, WO 2010/106051 and WO 2011/014438 can be cited.

LAG3 (lymphocyte activation gene 3, also known as CD223) belongs to the immunoglobulin (Ig) superfamily and contains 4 Ig-like extracellular domains. As an example of LAG3 antagonists, there may be cited the antibodies disclosed in WO 2010/019570.

HAVCR2 (hepatitis A virus cell receptor 2, also known as Tim-3, KIM-3; TIMD3; Tim-3; and TIMD-3) is a Th1-specific cell surface protein that belongs to the superfamily of immunoglobulins. HAVCR2 regulates macrophage activation and inhibits Th1-mediated auto and alloimmune responses, thereby promoting immune tolerance. As an example of HAVCR2 antagonists, there may be cited the antibodies disclosed in WO 2013 / 006490A.

ADORA2A (Adenosine A2A receptor, also known as A2aR, RDC8; or ADORA2) belongs to the guanine nucleotide-binding protein (protein G) receptor (GPCR) superfamily, which is subdivided into classes and subtypes. This protein plays an important role in many biological functions, such as circulation and heart rate, cerebral and renal blood flow, immune function, pain and sleep regulation. It has been implicated in pathophysiological conditions such as inflammatory diseases and neurodegenerative disorders.

Said antibody or fragment thereof and said conjugate are not linked.

Nucleic acids and vectors

As used herein, the term "polynucleotide" refers to RNA or DNA, preferably DNA.

Preferably, such a "polynucleotide" is operably linked to a gene expression sequence, which directs nucleic acid expression within a prokaryotic or eukaryotic cell, preferably within a eukaryotic cell. The "gene expression sequence" is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, that facilitates efficient transcription and translation of the immunocytokine nucleic acid to which it is operably linked. The gene expression sequence can be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter.

Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase, beta-actin promoter, muscle creatine kinase promoter, factor factor promoter human elongation and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, simian virus promoters (eg, VS40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), cytomegalovirus (C ^m V), Rous sarcoma virus (RSV), hepatitis B virus (HBV), the long terminal repeats (LTR) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes virus simple. Those of ordinary skill in the art are aware of other constitutive promoters.

Promoters useful as gene expression sequences also include inducible promoters. Inducible promoters are expressed in the presence of an induction agent. For example, metallothione in the promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

In general, the gene expression sequence will include, as necessary, 5 'non-transcribing and 5' non-translating sequences involved in the initiation of transcription and translation, respectively, such as a TATA box, cap sequence, CAAT sequence , and the like. Especially, such 5 'non-transcriptional sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably linked nucleic acid. Gene expression sequences optionally include enhancer sequences or 5 'activator sequences as desired. As used herein, the nucleic acid sequence encoding the immunocytokine of the invention and the gene expression sequence are said to be "operably linked" when they are covalently ligated in such a way as to position expression or transcription and / or translation of the sequence coding for the immunocytokine of the invention under the influence or control of the gene expression sequence.

Two DNA sequences are said to be operably linked if the induction of a promoter in the 5 'gene expression sequence results in transcription of the immunocytokine of the invention and if the nature of the binding between the two DNA sequences does not (1) results in the introduction of a frame shift mutation, (2) interferes with the ability of the promoter region to direct transcription of the immunocytokine of the invention, or (3) interferes with the ability of the corresponding RNA transcript which is going to translate into a protein. Thus, a gene expression sequence would be operably linked to a nucleic acid sequence encoding the immunocytokine of the invention if the gene expression sequence could transcribe that nucleic acid sequence so that the resulting transcript is translated into the desired polypeptide.

Advantageously, said nucleic acid sequence comprises an intron, since the previous mRNA molecules have often been shown to improve the production yields of recombinant molecules. Any intron sequence can be used, and as an example, the one disclosed in ZAGO et al. (Biotechnol. Appl. Biochem., Vol. 52 (Pt 3), pp .: 191-8, 2009) and in CAMPOS-DA-PAZ et al. (Mol. Biotechnol., Vol. 39 (2), pp .: 155-8, 2008).

The polynucleotide encoding the conjugate or the immunomodulatory antibody can be administered in vivo alone or in association with a vector.

In its broadest sense, a "vector" is any vehicle that can facilitate the transfer of the nucleic acid encoding the immunocytokine of the invention to cells. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the degree of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, cosmids, phagemids, episomes, artificial chromosomes, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by insertion or incorporation of the sequences. of immunocytokine nucleic acid.

Plasmid vectors are a preferred type of vector and have been extensively described in the art and are well known to those skilled in the art. See, for example, SANBROOK et al., "Molecular Cloning: A Laboratory Manual," second edition, Cold Spring Harbor Laboratory Press, 1989. Non-limiting examples of plasmids include pBR322, pUC18, pUC19, pRC / CMV, VS40, and pBlueScript. , and other plasmids are well known to those of ordinary skill in the art. Furthermore, plasmids can be custom designed using restriction enzymes and ligation reactions to remove and add specific DNA fragments.

Preferably, the vector can include selectable markers that are active on both bacterial and mammalian cells.

Pharmaceutical compositions and medical uses

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, or a primate, and most preferably a human.

The pharmaceutical composition of the invention can include one or more pharmaceutically acceptable carriers. The term "pharmaceutically acceptable" refers to compositions and molecular entities that are physiologically tolerable and do not normally cause allergic reactions or similar undesirable reactions, such as gastric distress, dizziness, and the like when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means that it may be approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term "carrier" refers to a solvent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.

In the context of the invention, the term "treat" or "treatment", as used herein, means to reverse, alleviate, inhibit the evolution of, or prevent the disorder or condition to which such term is applied, or one or more symptoms of such disorder or condition. The term "treating cancer" as used herein, means inhibition of the growth of cancer cells. Preferably, such treatment also leads to remission of tumor growth, that is, decrease in the size of a measurable tumor. Most preferably, such treatment leads to complete remission of the tumor.

The term "therapeutically effective amount" refers to an amount that is sufficient to inhibit the growth of cancer cells, preferably sufficient to induce remission of tumor growth. The administered doses can be adapted depending on various parameters, in particular depending on the mode of administration used, the relevant pathology, or alternatively the duration of treatment desired. Naturally, the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend on the condition to be treated, the severity of the condition, the age, weight and sex of the subject, etc. The effective dose ranges provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dose can be adjusted for each subject, as understood and determined by one of skill in the art, without undue experimentation.

In view of the remarkable efficacy of the combination of the invention, the skilled person may plan to use very small doses of both individual compounds to treat a subject.

As a non-limiting example, the immunosuppressive receptor antagonist antibody of the invention can be administered by injection at a dose of 500 pg / kg or less, preferably at a dose of 100 pg / kg or less, and most preferably at a dose of 50 pg / kg or less.

Preferably, said immunosuppressive receptor antagonist antibody can be administered by injection at a dose of at least 1 pg / kg of the subject, preferably of at least 5 pg / kg of the subject, and most preferably at a dose of at least 10 pg / kg of the subject.

As a non-limiting example, the conjugate of the invention can be administered by injection at a dose of 60 pg / kg or less, preferably at a dose of 10 pg / kg or less, and most preferably at a dose of 5 pg / kg or less.

Preferably, said conjugate can be administered by injection at a dose of at least 0.5 pg / kg of the subject, preferably at least 0.7 pg / kg of the subject, and most preferably at a dose of at least 0.8 pg / kg of the subject.

As an example, the administration steps may correspond to topical, oral, intranasal, intraocular, intravenous, intramuscular, intratumoral or subcutaneous administrations, and the like. These administration steps preferably correspond to injection. Thus, the conjugate, the immunomodulatory antibody or fragment thereof, the polynucleotide encoding such polypeptides, or the vectors comprising such polynucleotides are associated with carriers that are pharmaceutically acceptable for a formulation intended for injection. These can be, in particular, isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry compositions, especially lyophilized, which after the addition, as appropriate, of sterilized water or physiological saline, allow the constitution of injectable solutions. Suitable pharmaceutical carriers are described in EW Martin's "Remington's Pharmaceutical Sciences".

The conjugate, immunomodulatory antibody or fragment thereof, the polynucleotide encoding such polypeptides, or the vectors comprising such polynucleotides can be solubilized in a buffer or water or incorporated into emulsions, microemulsions, hydrogels (eg hydrogels based on triblock copolymers. PLGA-PEG-PLGA), in microspheres, in nanospheres, in microparticles, in nanoparticles (for example, microparticles of poly (lactic-co-glycolic acid) (for example, poly (lactic acid) (PLA); poly (acid lactide-coglycolic) (PLGA); polyglutamate microspheres, nanospheres, microparticles or nanoparticles), in liposomes, or other galenic formulations. In all cases, the formulation must be sterile and fluid to the degree of acceptable injectability. It must be stable in the manufacturing and storage conditions and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds can be prepared as free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropyl cellulose.

Dispersions in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils can also be prepared. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The conjugate, immunosuppressive receptor antagonist antibody or fragment thereof, the polynucleotide encoding such polypeptides, or the vectors comprising such invention polynucleotides can be formulated to give compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) that are formed with inorganic acids such as, for example, hydrochloric and phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Polypeptides can also be modified, by pegylation as an example, to increase their bioavailability.

Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of injectable compositions can be achieved through the use in the compositions of agents that delay absorption, for example, aluminum monostearate, gelatin, polyols, and covalent and non-covalent formulations that enhance half-life.

There are numerous causes of degradation or peptide instability, including hydrolysis and denaturation. Hydrophobic interaction can cause agglomeration of molecules together (i.e., aggregation). Stabilizers can be added to reduce or avoid such problems.

Stabilizers include cyclodextrin and derivatives thereof (see US Patent 5,730,969). Suitable preservatives such as sucrose, mannitol, sorbitol, trehalose, dextran, and glycerin can be added to stabilize the final formulation. A stabilizer selected from ionic and nonionic surfactants, D-glucose, D-galactose, D-xylose, D-galacturonic acid, trehalose, dextrans, hydroxyethyl starches, and mixtures thereof, may be added to the formulation. The addition of alkali metal salt or magnesium chloride can stabilize a peptide. The peptide can also be stabilized by contacting it with a saccharide selected from the group consisting of dextran, chondroitin sulfuric acid, starch, glycogen, dextrin, and alginic acid salt. Other sugars that may be added include monosaccharides, disaccharides, sugar alcohols, and mixtures thereof (eg, glucose, mannose, galactose, fructose, sucrose, maltose, lactose, mannitol, xylitol). Polyols can stabilize a peptide, and are water-miscible or water-soluble. Suitable polyols can be polyhydric alcohols, monosaccharides, and disaccharides including mannitol, glycerol, ethylene glycol, propylene glycol, trimethylglycol, vinylpyrrolidone, glucose, fructose, arabinose, mannose, maltose, sucrose, and polymers thereof. Various excipients can also stabilize peptides, including serum albumin, amino acids, heparin, fatty acids and phospholipids, surfactants, metals, polyols, reducing agents, metal chelating agents, polyvinylpyrrolidone, hydrolyzed gelatin, and ammonium sulfate.

In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences, and examples. However, the details of the examples are not intended to limit the invention.

Examples

1) PD1 / PD-L1 cancer model

BALB / C mice were injected subcutaneously into the right flank with CT26 tumor cells (2x105 / mouse). On day 9, when the tumors reached 20-30 mm2, mice were treated with 250 | ig / mouse anti-PD1 (BIOXCELL, clone RPM1-14, # BE0146) alone or isotype control (rat IgG2a; BIOXCELL, clone 2A3, # BE0089). Mice were injected anti-PD1 (a-PD1) or isotype control (2A3) on day 9, 12 and 15. From day 13 to day 37, mice were treated twice weekly with 2 | ig RLI CHO (1004-14p) or PBS in control groups. Tumors were measured three times per week with a caliper gauge and the tumor area was calculated as follows: length x width. Mice were sacrificed when tumor size reached 300 mm2 or ulcerated.

The protocol is presented in Figure 1. Mice bearing CT26 tumor received three 3 doses (250 pg / dose) of anti-PD1 or isotype control from day 9 to day 15. RLI treatment was injected (2 pg / dose) from day 13 to day 37 twice a week.

The CT26 tumor model expressed PD-L1 (ligand for PD1; (DURAISWAMY et al., Cancer Res., June 5, 2013) and was infiltrated by T cells that highly express the PD1 receptor. Figure 2 shows the elevated frequency of tumor-infiltrating CD8 + T cells co-expressing PD-1 and Tim-3 in model CT26 Stained and dissociated tumor of mouse bearing CT26 tumor were stained and analyzed by flow cytometry. Tim-3 co-expression and PD-1 in T cells CD8 + ^c + D3 shown in the spleen (left panel) and in the tumor (right panel).

Figure 3 shows the evolution of tumor size in CT26 tumor-bearing mice treated with anti-PD1 (aPD1) or control (2A3), RLI or control (PBS) or combining both treatments (aPD1 RLI) according to the protocol previously described. (A) Growth of the tumor in each group (n = 5 mice / group) (B) As in A., but from a second experiment. C. Percentage of mice with complete tumor remission in each group. (D-G). Tumor growth in each mouse (pooled from Experiment 1 and 2, n = 10) for the treated control group (D); for the independent group treated with RLI (F); for the independent group treated with aPD1 (F) and for the group treated with aPD1 RLI (F). Statistical tests were determined using 2-factor ANOVA. * p <0.05; *** p <0.001.

Figure 4 shows that the anti-PD1 / RLI combination treatment increases overall survival in mice. A. Overall survival in each treated group (n = 10 per group).

Therefore, the results show that the anti-PD1 + RLI combination treatment potentiates the antitumor effect compared to independent groups. In particular, RLI / aPD1 treatment increases the number of mice with complete tumor remission and overall survival (Figures 3C and 4). Surprisingly, no tumor-free mice treated with a combination of RLI and anti-PD1 that were re-exposed on day 145 after total remission with CT26 tumors showed no tumor growth, suggesting the persistence of protective antitumor immunity. (data not revealed). These results were confirmed in another series of experiments establishing that i) by itself, RLI had no effect on tumor growth when treatment started from D10 after tumor inoculation, ii) at that time, anti -PD1 enhanced survival but hardly induced complete remission (5.9%). The combination of anti-PD1 and RLI increased survival and complete remissions (30.4%), which were long-lasting remissions. This combination induced an effective anti-tumor memory immune response since the mice were still tumor-free 145 days after total remission and the re-exposure of cured mice never showed tumor recovery (data not shown).

BALB / C mice were injected subcutaneously into the right flank with CT26 tumor cells (2x105 / mouse). Mice received i.p. low-dose anti-PD1 (mBAT clone, 12 | ig / mouse) or control, 2 | ig RLI CHO (1004-15p) or control, or combination of both treatments on day 10. One week later (day 17) , mice receive a second injection of anti-PD1, RLI, or controls. Tumors were measured every other day with a caliper and the tumor area was calculated as follows: length x width. Mice were sacrificed when tumor size reached 300 mm2 or ulcerated. The protocol is presented in figure 5.

Figure 6 shows CT26 tumor growth in CT26 tumor bearing mice treated with two i.p. of low-dose anti-PD1 in combination with RLI. Statistical tests were determined using 2-factor ANOVA. *** p <0.001.

The results show that a low dose of anti-PD1 or RLI alone cannot support the inhibition of the growth of the primary subcutaneous tumor. However, the combination of low-dose anti-PD1 with RLI allowed inhibition of the tumor growth. This experiment confirmed that RLI could be synergized with anti-PD1 strategies to combat tumor growth even with a suboptimal dose of anti-PD1 treatment.

Finally, it appears that this low-dose combination results in a remarkable synergy that results in strong inhibition of tumor growth.

2) Metastatic melanoma

B16F10 tumors are implanted by injecting 3 x 104 B16F10 cells into the flank of C57BL / 6 mice, i.d. on day 0. i.p. with anti-CTLA-4 (clone: UC10-4F10-11 100 or clone: 9D9) or with the anti-PD-1 mAb (clone RPM1-14, No. BE0146) or with the anti-PD-L1 mAb ( clone: 10F.9G2) in combination or not with RLI CHO (2 pg / mouse) injected ip from day 7 to day 25 twice a week with 2 pg of RLI CHO. The dosage of anti-CTLA-4 or anti-PD-1 or anti-PD-L1 antibody by injection is 200 pg on day 3 plus 100 pg on days 6 and 9. Control groups received a corresponding dose of antibody isotype . Tumor incidence and size were monitored over time by physical examination.

3) Advanced lung cancer

The TC-1 tumor cell line was derived from primary lung epithelial cells of C57BL / 6 mice and is E6 / E7 human papillomavirus 16 (HPV-16) and cotransformed c-Ha-Ras. S.c. 105 TC-1 lung cancer cells in the upper dermis in the back of C57BL / 6 mice. Treatment begins on day 10 after tumor inoculation, which corresponds to when the tumors become clearly visible and palpable at a size of “15-20 mm2. The anti-CTLA-4 mAb (100 pg / mouse, clone: UC10-4F10-11 100 or clone: 9D9) or the anti-PD-I mAb (250 pg / mouse, clone RPM1-14, No. BE0146) o the anti-PD-L1 mAb (100 pg / mouse, clone: 10F.9G2) is provided at three unique time points (day 10, 14, 17) and RLI (2 pg / mouse) is provided on day 10, 13 and 18. Treatment with individual agents is compared against doublet combinations of each antibody with RLI, using tumor growth measured twice weekly as the end point. Control groups received a corresponding dose of antibody isotype.

4) Advanced bladder cancer

The MB49 tumor cell line originates from a carcinogen-induced tumor of bladder epithelial origin from male C57BL / 6 mice. S.c. 106 MB49 bladder cancer cells in the upper dermis in the back of C57BL / 6 mice. Treatment begins on day 6 after tumor inoculation, which corresponds to when the tumors become clearly visible and palpable at a size of “15 mm2. Anti-CTLA-4 mAb (100 pg / mouse, clone: UC10-4F10-11 100 or clone: 9D9) or anti-PD-1 mAb (100 pg / mouse, clone RPM1-14, # BE0146) or the anti-PD-L1 mAb (100 pg / mouse, clone: 10F.9G2) is provided at four unique time points (day 6, 9, 12) and RLI (2 pg / mouse) is provided on day 7, 8, 10, 11, 13, 14, and 16, 17. Treatment with individual agents is compared against doublet combinations of each antibody with RLI. Control groups received a corresponding dose of antibody isotype. Tumors were measured three times per week with a caliper gauge and the tumor area was calculated as follows: length x width. Mice were sacrificed when tumor size reached 300 mm2 or ulcerated.

5) breast cancer

BALB / c mice are inoculated with 5 x 104 4T1 breast cancer cells in the mammary gland on day 0. Treatment is started on day 10 after tumor inoculation which corresponds to when the tumors become clearly visible and palpable to a size of “15-20 mm2. The anti-CTLA-4 mAb (100 pg / mouse, clone: UC10-4F10-II 100 or clone: 9D9) or the anti-PD-1 mAb (250 pg / mouse, clone RPM1-14, No. BE0146) o the anti-PD-L1 mAb (200 pg / mouse, clone: 10F.9G2) is provided at four unique time points (day 10, 13, 16) and RLI (2 pg / mouse) is provided on day 10, 11, 13, 14, 16, 17, and 19, 20. Treatment with individual agents is compared against doublet combinations of each antibody with RLI. Control groups received a corresponding dose of antibody isotype. Tumors are measured three times per week with a caliper gauge and the tumor area is calculated as follows: length x width. Mice are sacrificed on day 27. Lung metastatic nodules are counted under a binocular microscope.

6) Ovarian cancer

The ID8-VEGF ovarian carcinoma cell line was previously developed from a mouse ovarian epithelial papillary serous adenocarcinoma cell line. C57BL / 6 mice are implanted subcutaneously in the right flank with 5 x 10 6 ID8 tumor cells. Treatment begins on day 10 after tumor inoculation, which corresponds to when the tumors become clearly visible and palpable at a size of “15-20 mm2. Anti-CTLA-4 mAb (100 pg / mouse, clone: UC10-4F10-11 100 or clone: 9D9) or anti-PD-1 mAb (250 pg / mouse, clone RPM1-14, # BE0146) o the anti-PD-L1 mAb (200 pg / mouse, clone: 10F.9G2) is provided at four unique time points (day 10, 13, 16) and RLI (2 pg / mouse) is provided on day 10, 11, 13, 14, 16, 17, and 19, 20. The

Claims (1)

Combined pharmaceutical composition comprising: a) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof having at least 10% of the activity of human IL-15 on the induction of proliferation of the kit225 cell line , and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to human IL-15 , a polynucleotide encoding therefor, or a vector comprising such a polynucleotide; and b) an antibody that antagonizes an immune pathway involved in inhibiting T-cell activation, or a fragment thereof that can react with the same antigen as its antibody counterpart, a polynucleotide encoding it, or a vector that it comprises such a polynucleotide, wherein said antibody is a PD-1 / PD-L1 / PD-L2 antagonist, wherein said antibody antagonizes either by binding to the PD-1 receptor or by binding to PD-L1 or PD-L2 ligand; wherein said conjugate and the antibody or fragment thereof are not linked. Pharmaceutical composition for use in treating cancer in a subject comprising (A) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof having at least 10% of the activity of human IL-15 on induction of cell line proliferation kit225, and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to IL-15 human, a polynucleotide encoding therefor, or a vector comprising such a polynucleotide, wherein the use comprises the simultaneous, separate or sequential administration of (a) said pharmaceutical composition and (b) an antibody that antagonizes an immune pathway involved in the inhibition of T-cell activation, or a fragment thereof that can reacting with the same antigen as its antibody counterpart, a polynucleotide encoding it, or a vector comprising such a polynucleotide, or (B) an antibody that antagonizes an immune pathway involved in the inhibition of T-cell activation, or a fragment thereof that can react with the same antigen as its antibody counterpart, a polynucleotide encoding it, or a vector comprising such a polynucleotide, wherein the use comprises the simultaneous, separate or sequential administration of (a) said pharmaceutical composition and (b) a conjugate comprising (i) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof that have at least 10% of the activity of human IL-15 on inducing proliferation of the kit225 cell line, and (ii) a polypeptide comprising the amino acid sequence of the sushi domain of IL- 15Ra or derivatives thereof having at least 10% of the binding activity of the sushi domain of human IL-15Ra to human IL-15, a polynucleotide encoding it, or a vector comprising a polynucl such an eotide, and wherein said antibody is a PD-1 / PD-L1 / PD-L2 antagonist, wherein said antibody antagonizes either by binding to the PD-1 receptor or by binding to the PD-L1 or PD-L2 ligand. Pharmaceutical composition for use in the treatment of cancer according to claim 2 or combined pharmaceutical composition according to claim 1, adapted for simultaneous, separate or sequential administration for use in the treatment of cancer in a subject. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 3, wherein: a) the conjugate is administered by injection at a dose of 60 pg / kg or less, preferably at a dose of 10 pg / kg or less and most preferably at a dose of 5 pg / kg or less; and b) the antibody that antagonizes an immune pathway involved in the inhibition of T cell activation, or a fragment thereof is administered by injection at a dose of 500 pg / kg or less, preferably at a dose of 100 pg / kg or less, and most preferably at a dose of 50 pg / kg or less. 5. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 4, wherein said antibody is a PD-1 / PD-L1 antagonist. 6. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 5, wherein said interleukin 15 derivative has at least 25% of the activity of human interleukin-15 on the induction of proliferation of the kit225 cell line, more preferably at least 50%, or it has an amino acid sequence having a percent identity of at least 92.5% with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferably of at least 96%, and more preferably of at least 98.5% or at least 99%. 7. A pharmaceutical composition or combined pharmaceutical composition according to any one of claims 1 to 6, wherein said sushi domain derivative of IL-15Ra has at least 25%, more preferably at least 50% of the binding activity of the Sushi domain of human IL-15Ra, or has an amino acid sequence that has a percentage identity of at least 92% with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID N ^o : 9, preferably at least 96%, and more preferably at least 98%. 8. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 7, wherein said antibody is selected from PD-1 / PD-L1 and PD-1 / PD antagonists. -L2, preferably nivolumab, Merck 3745, CT-01 1, lambrolizumab, AMP514, MDX-1 105 or YW243.55.S70. 9. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 8, wherein the polypeptides (i) and (ii) of the conjugate are covalently linked in a fusion protein . 10. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 9, wherein said interleukin 15 has the amino acid sequence SEQ ID NO: 1. 11. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 10, wherein the sushi domain of IL-15Ra has the amino acid sequence SEQ ID NO: 4. 12. Pharmaceutical composition for use in the treatment of cancer or combined pharmaceutical composition according to any one of claims 1 to 11, wherein the conjugate has the sequence SEQ ID NO: 16 or SEQ ID NO: 17.